Project Summary The goal of this project is to develop a light inducible CRISPR epigenetic editing tool to be used in specific reward neuron subtypes during cocaine self-administration and relapse. Epigenetic mechanisms, such as alterations in the accessibility of genes within their native chromatin structure induced by histone tail modifications, can underlie some of the persistent behaviors associated with drug abuse. Many studies use global overexpression or deletion of histone modifying enzymes in reward brain regions to alter these epigenetic processes during drug-mediated behaviors. Further, recent studies use zinc finger transcription factors to alter histone modifications at specific genes in reward brain regions during cocaine-mediated behaviors or other motivational behaviors. Such studies have provided critical information into these chromatin mediated processes in drug abuse. However, these tools lack temporal specificity which is greatly needed given the complex temporal behavioral dynamics occurring throughout the addictive process. We will move the field forward by developing an optically induced CRISPR mediated histone demethylation tool. We will use the CRY2/CIBN optical system in which dCas9 is fused to CIBN and the histone lysine demethylase 1A, KDM1A, is fused to CRY2, combined with gRNAs targeted to specific gene loci to generate a tool we term opto-CRISPR-KDM1A. This tool can be used to alter histone methylation at specific gene loci in the two nucleus accumbens projection neurons, the dopamine receptor 1 and dopamine receptor 2 expressing medium spiny neurons (D1-MSNs and D2-MSNs) due to their critical and often opposing role in behavioral responses to drugs of abuse. In this grant proposal we will alter lysine methylation at promoters and genes of the transcription factor early growth response 3 (Egr3) and its corepressor NGFI-A binding protein 2 (Nab2). We focus on these molecules because they are bidirectionally altered in D1-MSNs vs. D2-MSNs after repeated cocaine exposure. Further, Egr3 can bidirectionally regulate behavioral outcomes to cocaine through D1- MSNs vs. D2-MSNs. This allows an established set of predictions for behavioral outcomes during specific time points of cocaine self-administraiton and relapse when using the opto-CRISPR-KDM1A targeted to Egr3 and Nab2 promoters and genes in D1-MSNs vs. D2-MSNs. Once developed this tool can be used with multiple genes of interest by simple gRNA targeting to these genes. In the future, KDM1A can be swapped out for other histone modifying enzymes, DNA methylation enzymes, or transcription factors to generate many opto- CRISPR-epigenetic, activation, or interference tools with precise temporal specificity. Overall our studies will generate a new tool for epigenome editing with precise temporal regulation to be used in specific neuron subtypes during cocaine self-administration and relapse. This tool can be generalizable to many drug behavior paradigms and addiction related neuron subtypes to provide improved information into the temporal dynamics of histone lysine demethylation at specifc gene loci in drug abuse.